新城疫病毒TL1株L基因的克隆及其真核表达载体的构建

目的对新城疫病毒TL1株L基因进行克隆、序列分析及真核表达载体构建。方法根据GenBank登录的新城疫病毒L基因序列,设计了一对引物,用RT-PCR对内蒙古分离株TL1的L基因进行整体扩增。将扩增产物提纯后,克隆入pGEM-Teasy载体,再亚克隆至真核表达载体pCI-neo上,通过酶切、PCR鉴定及测序进行验证。结果测序拼接得出L基因的全序列长度为6704bp,该基因的ORF总长为6615bp,编码2204个氨基酸。与GenBank登陆的12株参考毒株比较L基因编码区全核苷酸序列和氨基酸序列,发现TL1株与鹅源新城疫NA-1株、ZJ1株和SF02株的同源性极高,说明四者亲缘关系较近。结论已成功构建L基因真核表达载体,为对该株病毒进行反向遗传操作以及有关功能基因组研究奠定了基础。

Cloning and Construction of Eukaryotic Expression Vector for L Gene of Newcastle Disease Virus TL1 Strain

Objective To clone the L gene of Newcastle disease virus(NDV)TL1 strain,analyze its sequence and construct its eukaryotic expression vector.Methods Design a pair of primers according to the NDV L gene sequence reported in GenBank and amplify L gene from the NDV TL1 strain isolated in Inner Mongolia,China by RT-PCR.Purify the amplified gene and clone into pGEM-T easy vector,then subclone to eukaryotic expression vector pCI-neo.Identify the constructed recombinant plasmid by restriction analysis,PCR and sequencing.Results The amplified L gene,at a full-length of 6 704 bp and a ORF length of 6 615 bp,encoded 2 204 amino acids.The nucleotide and amino acid sequences at encoding region of L gene were compared with those of 12 reference strains in GenBank,and the result showed high homologies of TL1 strain to the NA-1,ZJ1 and SF02 strains of NDV from goose origin.ConclusionThe eukaryotic expression vector for L gene of NDV TL1 strain was successfully constructed,which laid a foundation of study on reverse genetics and functional genome of TL1 strain.